Project Summary Oxytocin (OT) regulates many aspects of social behavior, including parental nurturing, social information processing, and social attachment. OT is thought to influence social behaviors by enhancing the salience and reinforcing value of social stimuli. Intranasal administration of OT in humans increases attention to social cues, emotion detection and socially reinforced learning. Intranasal OT enhances some aspects of social functioning in individuals with autism spectrum disorder (ASD), and the OT system is a leading pharmacological target for enhancing social function in ASD. Single nucleotide polymorphisms (SNPs) in noncoding regions of the human OT receptor gene (OXTR) are associated with core symptoms of ASD, altered brain activity patterns, and diagnosis of ASD. However, human gene association studies do not provide insights into how polymorphisms in OXTR lead to variation in brain function or social behavior. The socially monogamous prairie vole is an ideal model organism to explore the precise molecular mechanisms by which variation in the OXTR gene can lead to alterations in brain phenotype, and downstream social behaviors. OXTR signaling in the nucleus accumbans (NAcc) is critically involved in alloparental nurturing and social bond formation in prairie voles. There is remarkable individual variation in the density of OXTR in the NAcc of prairie voles that is associated with variation in social behavior and with resilience to early life social neglect. A set of 14 SNPs in the prairie vole OXTR gene (Oxtr) explains up to 80% of the variation in OXTR density in the NAcc, but not in other brain areas. The goal of this proposal is to use prairie voles to explore how variation in Oxtr influences molecular processes and brain phenotypes that are part of machinery hypothesized to affect downstream behavioral phenotypes. Since the 14 SNPs strongly associated with NAcc OXTR density were in perfect linkage disequilibrium in the samples studied thus far, it is not possible to determine which of those SNPs is most likely to be influencing Oxtr expression. The first Aim will examine the association of the candidate SNPs with NAcc OXTR density in 230 genetically diverse prairie voles to identify the SNPs most strongly associated with OXTR density in the NAcc. The second Aim will examine the influence of the candidate SNPs and their associated molecular phenotype on coordinated brain activity during sociosexual interactions. The third Aim will use chromatin immunoprecipitation (ChIP) to characterize the regulatory landscape of the Oxtr in NAcc and other brain regions in order to further refine the list of SNPs most likely to be influencing brain phenotype. Finally, the CRISPR/Cas9 genome editing system will be used to edit the SNPs most likely to be functional in order to identify those with the greatest influence on OXTR density in the NAcc. Characterization of the regulatory status of the SNP most likely to be influencing OXTR expression and the consequences on brain function will provide important insights that will guide future human genetic studies investigating the potential influence of OXTR SNPs on brain phenotype, social cognition and psychopathology.